A method for producing (S)—N-protected-propargylglycine of the following formula (2), wherein the method comprises asymmetrically hydrolyzing an N-protected-propargylglycine ester of the following formula (1) by using an asymmetric hydrolysis enzyme or a cultured substance of a microorganism having an ability of producing this enzyme or a treated substance thereof. The hydrolysis enzyme is obtained from a microorganism selected from the group consisting of Thermomyces genus, Aspergillus genus, Rhizopus genus, Penicillium genus, Pseudomonas genus, Humicola genus, Burkholderia genus, Candida genus Bacillus genus and Streptomyces genus.